There are a large variety of small polynucleotides, both naturally occurring and synthetic, which are of scientific or commercial interest. Exemplary small polynucleotides include microRNAs, snoRNAs, short interfering RNAs (natural or synthetic), guide RNAs, nucleolar RNAs, ribosomal RNAs, tRNAs as well as small antisense DNAs or small polynucleotide degradation products. Of particular interest are microRNAs (miRNAs), naturally occurring, single stranded polyribonucleotides (polyRNAs) of between 18 and 24 RNA residues, which are derived from a longer, naturally occurring noncoding eukaryotic precursor RNA transcript (usually having a ‘hairpin’ configuration), and miRNAs play a significant role in cellular developmental and differentiation pathways. Consequently, there have been considerable efforts made to understand and characterize the temporal, spatial and cellular expression levels and patterns of expression of miRNAs to ascertain their precise role in cellular development and differentiation in both normal and disease states.
miRNAs are currently studied by, first, obtaining the total RNA from a sample. Next, the total RNA is fractionated into subpopulations by gel electrophoresis or by chromatographic fractionation and size selective elution. Then, the appropriate section of the gel is cut, and the 18-24 RNAs are eluted from the gel, or the eluted fraction containing single stranded RNA's in the size range of 18-24 ribonucleotides is collected, usually the RNA fraction of less than 500-200 nucleotides in length. Next, the RNAs are isolated by precipitation and the miRNAs are characterized.
However, these methods are disadvantageous because they do not work well when the amount of sample is small, such as samples from tumor tissue or biopsy material. Further, characterization of the miRNAs isolated by present methods usually comprises a several step amplification procedure followed by detection, quantitation, cloning and sequencing. Because of the large number of steps in these processes, and the notorious inefficiencies associated with the repeated purification, isolation and identification of miRNAs, it is time consuming, relatively expensive, requires relatively large amounts of material and is not fully representative of the population of miRNAs expressed within a sample, such as within a tumor, or those miRNAs expressed in low abundance. Additionally, the methods are not specific to isolating and identifying miRNAs, and often isolate and identify siRNA, tRNA, 5S/5.8SrRNA and degraded RNA from additional cellular RNAs.
Therefore, there is the need for an improved method for isolation and identification of miRNAs, other small regulatory RNAs and short interfering RNAs (siRNAs) that is not associated with these disadvantages.